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Advances in Bioscience Education Summer Workshop Fluorescence and Electron Microscopy June 26 - 29, 2007 Biological Electron Microscope Facility Pacific Biosciences Research Center University of Hawai’i at Manoa What is a Microscope? A tool that magnifies and improves resolution of the components of a structure Has three components: sources of illumination, a magnifying system, detectors. Sources of Illumination Light microscopes use a beam of light for illumination and include fluorescence and confocal microscopes Electron microscopes use electrons as a source of illumination and include transmission and scanning electron microscopes. Light and Electron Microscopes Lenses are used to control a beam of illumination, magnify, and direct an image to a detector Images and pictures are your data! Epifluorescence Microscopy Common Fluorescence Applications Localize/identify specific organelles Detect live cells vs. dead cells, necrotic vs. apoptotic cells Determine cell membrane permeability Localize antigen-specific molecules Multiple labeling Laser Scanning Confocal Microscope Better resolution Serial optical sections can be collected from thick specimens Live or fixed cell and tissue imaging Laser Scanning Confocal Microscopy Drosophila eye Plant Protoplast Photos courtesy of Gregg Meada & Dr. Gert DeCouet, UHM And Dr. Chris Yuen and Dr. David Christopher Epifluorescence vs. Confocal Sample courtesy Gregg Meada & Dr. Gert DeCouet, UHM Scanning Electron Microscopy (SEM) View outer surface Coat specimen with gold No sectioning High Mag (40x to 300,000x) High resolution (better than 2 nm) SEM Images Transmission Electron Microscopy (TEM) View inside cell via sections magnification 120,000 X 50,000X Conventional TEM Micrographs Bacteria in cell Apoptosis Skin Collagen Chloroplast Virus in cell Ultra-microtomy Ultrathin (60-90 nm) sectioning of resinembedded specimens Several brands/models available Cryotechniques Ultrarapid cryofixation Metal mirror impact Liquid propane plunge Freeze fracture with Balzers 400T Cryosubstitution Cryoultramicrotomy – Ultrathin frozen sections (primarily for antibody labeling) Immunolocalization LM Fluor/confocal TEM SEM with backscatter detector Approaches to Immunolabeling Direct Method: Primary antibody contains label Indirect Method: Primary antibody followed by labeled secondary antibody Amplified Method: Methods to add more reporter to labeled site Two-step Indirect Method for Immunolabeling Fluorescentconjugated secondary antibody attaches to primary antibody that is bound to antigen Immunolabeling for Transmission Electron Microscopy Normally do Two-Step Method Primary antibody applied followed by colloidal gold-labeled secondary antibody May also be enhanced with silver Colloidal Gold Immunolabeling for TEM Colloidal gold of defined sizes, e.g., 5 nm, 10 nm, 20 nm, easily conjugated to antibodies Results in small, round, electron-dense label easily detected with EM Can be enhanced after labeling to enlarge size for LM or EM Double-labeling Method Use primary antibodies derived from different animals (e.g., one mouse antibody and one rabbit antibody) Then use two different secondary antibodies conjugated with different sized gold particles Preparation of Biological Specimens for Immunolabeling Preserve tissue as closely as possible to its natural state while at the same time maintaining the ability of the antigen to react with the antibody Chemical fixation OR Cryofixation Chemical Fixation Antigenic sites are easily denatured or masked during chemical fixation Glutaraldehyde gives good fixation but may mask antigens, plus it is fluorescent Paraformaldehyde often better choice, but results in poor morphology , especially for electron microscopy May use e.g., 4% paraformaldehyde with 0.5% glutaraldehyde as a good compromise Embedding Dehydrated tissue is embedded in a plastic resin to make it easier to cut thin sections Steps in Labeling of Sections Chemical fixation Dehydration, infiltration, embedding and sectioning Blocking Incubation with primary antibody Washing Incubation with secondary antibody congugated with reporter (fluorescent probe, colloidal gold) Washing, optional counterstaining Mount and view Controls! Controls! Controls! Omit primary antibody Irrelevant primary antibody Pre-immune serum Perform positive control Check for autofluorescence Check for non-specific labeling Dilution series Light Microscopes Light Path in Fluorescence Light delivered through excitation filter and then objective lens to specimen where it is absorbed; emitted light goes back through objective lens through barrier filter and emission filter and then to detector. Fluorescence Light beam excites the fluorochrome, raising it to a higher energy state, As it falls back to it’s original state, it releases energy in the form of a light of lower E and longer wavelength than original beam of light Primary Ab = PDI secondary Ab = Alexafluor Blue light = exciting beam green and red light emitted And use them to your advantage! Green is label; orange-red is autofluorescence Acts as counterstain Know Your Artifacts Autofluorescence Fluorescence Fluorochromes are excited by specific wavelengths of light and emit specific wavelengths of a lower energy (longer wavelength) Filter Cubes for Fluorescence Filter cubes generally have an excitation filter, a dichroic element, and an emission filter The elements of a cube are selected for the excitation and fluorescence detection desired Choose Fluorochrome/Filter Combos Laser Scanning Confocal Microscopy Fluorescence technique Uses laser light for excitation Improves image resolution over conventional fluorescence techniques Optically removes out-of-focus light and detects only signal from focal plane Can construct an in-focus image of considerable depth from a stack of images taken from different focal planes of a thick specimen Can then make a 3-D image that can be tilted, rotated, and sliced Principal Light Pathway in Confocal Microscopy Laser light is scanned pixel by pixel across the sample through the objective lens Fluorescent light is reflected back through the objective and filters (dichroic mirrors) Adjustable pinhole apertures for PMTs eliminate out-offocus flare Image is detected by photomultiplier(s) and digitized on computer TEM Transmission Electron Microscope Illumination source is beam of electrons from tungsten wire Electromagnetic lenses perform same function as glass lenses in LM Higher resolution and higher magnification of thin specimens Specimen Preparation for TEM Chemical fixation with buffered glutaraldehyde Or 4% paraformaldehyde with >1% glutaraldehyde Postfixation with osmium tetroxide Or not, or with subsequent removal from sections Dehydration and infiltration with liquid epoxy or acrylic resin Polymerization of hard blocks by heat or UV Ultramicrotomy – 60-80nm sections Labeling and/or staining View with TEM High pressure freezing: Plant tissue is flash frozen in a pressure bomb -197 C Water in the tissue is replaced with acetone over 5 day period Acetone saturated tissue is embedded in resin Resin is cut in thin sections, 80 nm thick Add antibodies - immunolabeling Look under Electron microscope Very Wrinkled Chloroplast Carnage Pretty bad fixation 2nd time: stainings were done poorly, but there is hope… Back to the drawing board to start over. But what to correct? What to do different? Will it improve? Despite mistakes, keep moving forward and ignore doubt and negativism that comes with pressure. 3rd time A charm Excellent preservation And Immunolabeling the 3rd TIME HIGH MAG RE-search Not search Must be repeated Research time is spent: 70% trouble-shooting 15% success 15% communicating success. ROOT HOOK-o-PLASM PDI in Vacuole CNGC in Golgi Apparatus g 200 nm PDI in Golgi Apparatus c G 200 nm Dividing mitochondria Channel located to the plasma membrane Channel located to the plasma membrane -plasmolysis We learn more from mistakes than successes…